Dataanalysis version 5

LC-MS analyses were performed with an ESI-QTOF-MS system (Impact II, Bruker, Billerica, MA, USA) coupled with a liquid chromatography system (1290 Infinity, Agilent) and equipped with a reversed-phase Agilent Zorbax SB-C18 column (2.1 mm × 50 mm, 1.8 µm). Mobile phases were water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). The following gradient elution method was used: 0 to 95% B from 0 to 45 min, a 95% B plateau for 10 min, and a return to initial conditions with equilibration for 10 min. The column temperature was set at 40 °C, and the flow rate was 0.25 mL/min. The samples were kept in an autosampler at 10 °C. Each sample was injected at a volume of 10 μL. The operating parameters of the mass spectrometer were as follows: negative spray voltage at 3500 V, dry temperature at 200 °C, dry gas flow at 8 L/min, and nebulizer at 1 bar. The data were collected with a mass range from 50 to 2500 m/z with an acquisition rate of 8 Hz and stepping. The Auto MS/MS mode was used to obtain fragment ions. The instrument was calibrated using a Tune mix standard solution before the runs, and, at the beginning of each run, this solution was injected as an internal calibrant for MS spectrum calibration. The LC-MS-acquired data were processed using DataAnalysis Version 5.2 software (Bruker) and Metaboscape Version 2023b software (Bruker).

LC-MS analyses were performed with an ESI-QTOF-MS system (Impact II, Bruker, Billerica, MA, USA) coupled with a liquid chromatography system (1290 Infinity, Agilent) and equipped with a reversed-phase InertSustain AQ-C18 column (100 × 2.1 mm; 3 µm) GL Sciences Inc. Mobile phases were water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). The following gradient elution method was used: 5% B from 0 to 1 min, 5−60% B from 1 to 12 min, a 60% B plateau for 12 to 16 min, 60−80% B from 16 to 20 min, an 80% B plateau for 3 min and a return to initial conditions with equilibration for 6 min. The column temperature was set at 30 °C, and the flow rate was 0.4 mL/min. The samples were kept in an autosampler at 4 °C. Each sample was injected at a volume of 10 μL. The operating parameters of the mass spectrometer were as follows: positive spray voltage at 4000 V, dry temperature at 250 °C, dry gas flow at 10 L/min and nebulizer at 3 bar. The data were collected with a mass range from 50 to 1000 m/z. The instrument was calibrated using a 10 mM sodium formate solution before the runs, and, at the beginning of each run, this solution was injected as an internal calibrant for MS spectrum calibration. The LC-MS acquired data were processed using DataAnalysis Version 5.2 software (Bruker) to extract the mass spectra from the sample raw data.